The invention relates to the use of poly (ADP-ribose) polymerase (PARP) proteins, particularly mutant PARP proteins or parts thereof, and genes encoding the same, to produce eukaryotic cells and organisms, particularly plant cells and plants, with modified programmed cell death. Eukaryotic cells and organism, particularly plant cells and plants, are provided wherein either in at least part of the cells, preferably selected cells, the programmed cell death (PCD) is provoked, or wherein, on the contrary, PCD of the cells or of at least part of the cells in an organism is inhibited, by modulation of the level or activity of PARP proteins in those cells. The invention also relates to eukaryotic cells and organisms, particularly plant cells and plants, expressing such genes.
Programmed cell death (PCD) is a physiological cell death process involved in the elimination of selected cells both in animals and in plants during developmental processes or in response to environmental cues (for a review see Ellis et al. 1991; Pennell and Lamb, 1997). The disassembly of cells undergoing PCD is morphologically accompanied by condensation, shrinkage and fragmentation of the cytoplasm and nucleus, often into small sealed packets (Cohen 1993, Wang et al. 1996). Biochemically, PCD is characterized by fragmentation of the nuclear DNA into generally about 50 kb fragments representing oligonucleosomes, as well as the induction of cysteine proteinases and endonucleases. The fragmentation of the DNA can be detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) of DNA 3xe2x80x2-OH groups in sections of cells. (Gavrieli et al. 1992). Cell death by PCD is clearly distinct from cell death by necrosis, the latter involving cell swelling, lysis and leakage of the cell contents.
In animals, PCD is involved in the elimination or death of unwanted cells such as tadpole tail cells at metamorphosis, cells between developing digits in vertebrates, overproduced vertebrate neurons, cells during cell specialization such as keratocytes etc. Damaged cells, which are no longer able to function properly, can also be eliminated by PCD, preventing them from multiplying and/or spreading. PCD, or the lack thereof, has also been involved in a number of pathological conditions in humans (AIDS, Alzheimer""s disease, Huntington""s disease, Lou Gehrig""s disease, cancers).
In plants, PCD has been demonstrated or is believed to be involved in a number of developmental processes such as e.g., removal of the suspensor cells during the development of an embryo, the elimination of aleurone cells after germination of monocotyledonous seeds; the elimination of the root cap cells after seed germination and seedling growth; cell death during cell specialization as seen in development of xylem tracheary element or trichomes, or floral organ aborting in unisexual flowers. Also the formation of aerochyma in roots under hypoxic conditions and the formation of leaf lobes or perforations in some plants seem to involve PCD. Large scale cell death in plants occurs during upon senescence of leaves or other organs. The hypersensitive response in plants, in other words the rapid cell death occurring at the site of entry of an avirulent pathogen leading to a restricted lesion, is an another example of PCD in response to an environmental cue.
Animal or plant cells dying in suspension cultures, particularly in low-density cell suspension cultures, also demonstrate the characteristics of PCD.
An enzyme which has been implied to be involved in PCD or apoptosis is poly(ADP-ribose) polymerase. Poly(ADP-ribose) polymerase (PARP), also known as poly(ADP-ribose) transferase (ADPRT) (EC 2.4.2.30), is a nuclear enzyme found in most eukaryotes, including vertebrates, arthropods, molluscs, slime moulds, dinoflagellates, fungi and other low eukaryotes with the exception of yeast. The enzymatic activity has also been demonstrated in a number of plants (Payne et al., 1976; Willmitzer and Wagner, 1982; Chen et al., 1994; O""Farrell, 1995).
PARP catalyzes the transfer of an ADP-ribose moiety derived from NAD+, mainly to the carboxyl group of a glutamic acid residue in the target protein, and subsequent ADP-ribose polymerization. The major target protein is PARP itself, but also histones, high mobility group chromosomal proteins, a topoisomerase, endonucleases and DNA polymerases have been shown to be subject to this modification.
The PARP protein from animals is a nuclear protein of 113-120 kDa, abundant in most cell types, that consist of three major functional domains: an amino-terminal DNA-binding domain containing two Zn-finger domains, a carboxy-terminal catalytic domain, and an internal domain which is automodified (de Murcia and Mxc3xa9nissier de Murcia, 1994; Kameshita et al., 1984; Lindahl et al., 1995). The enzymatic activity in vitro is greatly increased upon binding to single-strand breaks in DNA. The in vivo activity is induced by conditions that eventually result in DNA breaks (Alvarez-Gonzalez and Althaus, 1989; Ikejima et al., 1990). Automodification of the central domain apparently serves as a negative feedback regulation of PARP.
PARP activity in plant cells was first demonstrated by examining the incorporation of 3H from labelled NAD+ into the nuclei of root tip cells (Payne et al., 1976; Willmitzer and Wagner, 1982). The enzymatic activity was also partially purified from maize seedlings and found to be associated with a protein of an apparent molecular mass of 113 kDa, suggesting that the plant PARP might be similar to the enzyme from animals (Chen et al., 1994; O""Farrell, 1995).
cDNAs corresponding to PARP proteins have isolated from several species including mammals, chicken, Xenopus, insects and Caenorhabditis elegans. Chen et al. (1994) have reported PARP activity in maize nuclei and associated this enzymatic activity with the presence of an approximately 114 kDa protein present in an extract of maize nuclei. O""Farrel (1995) reported that RT-PCR-amplification on RNA isolated from maize (using degenerate primers based on the most highly conserved sequences) resulted in a 300 bp fragment, showing 60% identity at the amino acid level with the human PARP protein. Lepiniec et al. (1995) have isolated and cloned a full length cDNA from Arabidopsis thaliana encoding a 72 kDa protein with high similarity to the catalytic domain of vertebrate PARP. The N-terminal domain of the protein does not reveal any sequence similarity with the corresponding domain of PARP from vertebrates but is composed of four stretches of amino acids (named A1, A2, B and C) showing similarity to the N-terminus of a number of nuclear and DNA binding proteins. The predicted secondary structure of A1 and A2 was a helix-loop-helix structure.
The Genbank database contains the sequences of two cDNAS from Zea mays for which the amino acid sequence of the translation products has either homology to the conventional PARP proteins (AJ222589) or to the non-conventional PARP proteins, as identified in Arabidopsis (AJ222588)
The function(s) of PARP and poly-ADP ribosylation in eukaryotic cells is (are) not completely clear. PARP is involved or believed to be involved either directly or indirectly in a number of cellular processes such as DNA repair, replication and recombination, in cell division and cell differentiation or in the signalling pathways that sense alterations in the integrity of the genome. As PARP activity may significantly reduce the cellular NAD+ pool, it has also been suggested that the enzyme may play a critical role in programmed cell death (Heller et al., 1995; Zhang et al., 1994). Further, it has been suggested that nicotinamide resulting from NAD+ hydrolysis or the products of the turn-over of poly-ADP-ribose by poly-ADP-ribose glycohydrolase may be stress response signals in eukaryotes.
The information currently available on the biological function of plant PARP has come from experiments involving PARP inhibitors suggesting an in vivo role in the prevention of homologous recombination at sites of DNA damage as rates of homologous intrachromosomal recombination in tobacco are increased after application of 3-aminobenzamide (3ABA) (Puchta et al., 1995). Furthermore, application of PARP inhibitors, such as 3ABA, nicotinamide, and 6(5H)-phenasthridinone, to differentiating cells of Zinnia or of Helianthus tuberosum has been shown to prevent development of tracheary elements (Hawkins and Phillips, 1983; Phillips and Hawkins, 1985; Shoji et al., 1997; Sugiyama et al., 1995), which is considered to be an example of programmed cell death in plants.
PCT application WO97/06267 describes the use of PARP inhibitors to improve the transformation (qualitatively or quantitatively) of eukaryotic cells, particularly plant cells.
Lazebnik et al. (1994) identified a protease with properties similar to the interleukin 1-xcex2-converting enzyme capable of cleaving PARP, which is an early event in apoptosis of animal cells.
Kuepper et al. (1990) and Molinette et al. (1993) have described the overproduction of the 46 kDa human PARP DNA-binding domain and various mutant forms thereof, in transfected CV-1 monkey cells or human fibroblasts and have demonstrated the trans-dominant inhibition of resident PARP activity and the consequent block of base excision DNA repair in these cells.
Ding et al. (1992), and Smulson et al. (1995) have described depletion of PARP by antisense RNA expression in mammalian cells and observed a delay in DNA strand break joining, and inhibition of differentiation of 3T3-L1 preadipocytes.
Mxc3xa9nissier de Murcia et al., (1997) and Wang et al. (1995, 1997) have generated transgenic xe2x80x9cknock-outxe2x80x9d mice mutated in the PARP gene, indicating that PARP is not an essential protein. Cells of PARP-deficient mice are, however, more sensitive to DNA damage and differ from normal cells of animals in some aspects of induced cell death (Heller et al., 1995).
The invention provides a method for modulating programmed cell death in a eukaryotic cell, comprising reducing the functional level of the total PARP activity in a eukaryotic cell using the nucleotide sequence of a PARP gene of the ZAP class, and the nucleotide sequence of a PARP gene of the NAP class, preferably to reduce expression of the endogeneous PARP genes, to reduce the apparent activity of the proteins encoded by the endogenous PARP genes or to alter the nucleotide sequence of the endogenous PARP genes.
The invention also provides a method for modulating programmed cell death in a eukaryotic cell, comprising introducing a first and a second PCD modulating chimeric gene in a eukaryotic cell, preferably a plant cell, wherein the first PCD modulating chimeric gene comprises the following operably linked DNA regions: a promoter, operative in a eukaryotic cell; a DNA region, which when transcribed yields a RNA molecule which is either capable of reducing the functional level of a Zn-finger containing PARP protein of the ZAP class; or is capable of being translated into a peptide or protein which when expressed reduces the functional level of a PARP protein of ZAP class and a DNA region involved in transcription termination and polyadenylation
and wherein the second PCD modulating chimeric gene comprises the following operably linked DNA regions:a promoter, operative in the eukaryotic cell; a DNA region, which when transcribed yields a RNA molecule which is either capable of reducing the functional level of a PARP protein of the NAP class; or capable of being translated into a peptide or protein which when expressed reduces the functional level of a PARP protein of the NAP class, and a DNA region involved in transcription termination and polyadenylation; and wherein the total apparent PARP activity in the eukaryotic cell is reduced significantly, (preferably the total apparent PARP activity is reduced from about 75% to about 90% of the normal apparent PARP activity in the eukaryotic cell, and the eukaryotic cell is protected against programmed cell death) or almost completely (preferably the total apparent PARP activity is reduced from about 90% to about 100% of the normal apparent PARP activity in the eukaryotic cell, and the cell is killed by programmed cell death).
Preferably the first transcribed DNA region or the second transcribed DNA region or both, comprise a nucleotide sequence of at least about 100 nucleotides with 75% identity to the sense DNA strand of an endogenous PARP gene of the ZAP or the NAP class, and encode a sense RNA molecule is capable of reducing the expression of the endogenous PARP gene of the ZAP or the NAP class.
In an alternative method for modulating programmed cell death, provided by the invention, the first transcribed DNA region or the second transcribed DNA region or both, comprise a nucleotide sequence of at least about 100 nucleotides with 75% identity to the complement of the sense DNA strand of an endogenous PARP gene of the ZAP or the NAP class, and encode RNA molecule is capable of reducing the expression of said endogenous PARP gene of the ZAP or the NAP class.
In yet an alternative method for modulating programmed cell death, provided by the invention, the first and/or second transcribed DNA region encodes a RNA molecule comprising a sense nucleotide sequence of at least about 100 nucleotides with 75% identity to the mRNA resulting from transcription of an endogenous PARP gene of the ZAP or the NAP class and the RNA molecule further comprising an antisense nucleotide sequence of at least about 100 nucleotides with 75% identity to the complement of the mRNA resulting from transcription of the endogenous PARP gene of the ZAP or the NAP class, wherein the sense and antisense nucleotide sequence are capable of forming a double stranded RNA region, and wherein that RNA molecule is capable of reducing the expression of the endogenous PARP gene of the ZAP or the NAP class.
In a further alternative method for modulating programmed cell death, provided by the invention, the first and/or second transcribed DNA region encodes a dominant negative PARP mutant capable of reducing the apparent activity of the PARP protein encoded by an endogenous PARP gene of the ZAP or the NAP class, preferably comprising amino acid sequence selected from the amino acid sequence of SEQ ID No 4 from amino acid 1 to 159 or the amino acid sequence of SEQ ID No 6 from amino acid 1 to 138 or comprising an amino acid sequence selected from the amino acid sequence of SEQ ID No 2 from amino acid 1 to 370, the amino acid sequence of SEQ ID No 11 from amino acid 1 to 98, or the amino acid sequence of SEQ ID No 2 from amino acid 1 to 370 wherein the amino acid sequence from amino acid 1 to 88 is replaced by the amino acid sequence of SEQ ID No 11.
The promoter of the first and second chimeric PCD modulating genes, or both, may be a tissue specific or inducible promoter such as a promoter is selected from a fungus-responsive promoter, a nematode-responsive promoter, an anther-selective promoter, a stigma-selective promoter, a dehiscence-zone selective promoter.
The invention also provides a method for modulating programmed cell death in a plant cell, comprising introduction of a PCD modulating chimeric gene in said plant cell, wherein the PCD modulating chimeric gene comprises the following operably linked DNA regions: a plant-expressible promoter, a DNA region, which when transcribed yields a RNA molecule, which is either capable of reducing the expression of endogenous PARP genes; or is capable of being translated into a peptide or protein which when expressed reduces the apparent PARP activity in the plant cell, and a DNA region involved in transcription termination and polyadenylation, wherein the total apparent PARP activity in the plant cell is reduced from about 75% to about 100% of the normal apparent PARP activity in the plant cell.
It is another objective of the invention to provide the first and second chimeric PCD modulating gene as well as a eucaryotic cell, particularly a plant cell comprising the first and second chimeric PCD modulating gene and non-human eukaryotic organisms, particularly plants comprising such cells.
Finally, the invention also provides an isolated DNA sequence comprising the nucleotide sequence of SEQ ID No 1 from the nucleotide at position 113 to the nucleotide at position 3022, an isolated DNA sequence comprising the nucleotide sequence of SEQ ID No 10 from the nucleotide at position 81 to the nucleotide at position 3020 and an isolated DNA sequence comprising the nucleotide sequence of SEQ ID No 3 from the nucleotide at position 107 to the nucleotide at position 2068.